Method of elicting or inducing an immune response

ABSTRACT

A method for eliciting or inducing an immune response in a human or animal subject, comprises administering to said subject a composition comprising an antigen and an adjuvant, wherein the composition is administered to the subject by the intra-lung route.

FIELD OF THE INVENTION

This invention relates to a method of eliciting or inducing an immuneresponse in a subject, particularly a human subject, and moreparticularly to a method which utilises a needle-less route ofadministration, thereby providing an alternative to the more traditionalinjection routes for administration of vaccines such as the subcutaneousand intramuscular routes.

BACKGROUND OF THE INVENTION

Over many years, various attempts have been made to utilise therespiratory tract as a means to deliver non-living vaccine antigens.This route was seen as conferring advantages in terms of vaccineacceptability (avoiding “needle phobia”), the opportunity to inducelocal immune responses and the potential to induce responses at distantmucosal sites given the assumed unity of the mucosal immune system. Muchattention has been given to delivering vaccines by the intranasal routein humans and animals. Results to date indicate that this route requiresvery high antigen doses and/or the use of specialised deliverytechnologies to ensure antigen uptake and immune induction.

Similarly, delivery of vaccines via the intra-lung or pulmonary routehas in the past generally required specialised delivery techniques, suchas microencapsulation (see, for example, Oya Alpa et al., 2005), inorder to optimise immune responses.

In work leading to the present invention, the inventors have observedthat delivery of vaccines by the intranasal route is highly inefficient,inducing poor local immunity even at high antigen doses. Accordingly,the inventors have investigated an alternative route of vaccineadministration which retains the advantage of avoiding “needle phobia”.

Surprisingly, the inventors have demonstrated the superiority of vaccinedelivery to the lung over that by the intranasal route as a means ofinducing immune responses. They have also demonstrated that strongsystemic immune responses can be induced using very small quantities ofantigen when delivered with adjuvant via the lung. In particular, theinventors have shown that a straightforward vaccine compositioncombining antigen and adjuvant induces strong systemic immune responseson intra-lung delivery without the need for specialised uptaketechnologies. Significantly, it has been shown that intra-lung deliveryresults in lung mucosal immune responses, which can be as much as onehundred-fold (100×) greater than that induced by conventional parenteralimmunisation, even when very small quantities of antigen are deliveredwith adjuvant via the lung.

Griffith et al (1997) describe the intratracheal delivery of ricintoxoid formulated either in liposomes, with aluminium hydroxide or inPBS. The liposome formulated group showed best protection; aluminiumhydroxide did not improve protection over PBS. WO 2005/110379 describesthe pulmonary delivery of a particulate malaria vaccine. The formulationwas delivered as a particulate formulation and permitted sustainedrelease of antigen.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and or variations suchas “comprises” or “comprising”, will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

SUMMARY OF THE INVENTION

The present invention provides a method for eliciting or inducing animmune response in a human or animal subject, which comprisesadministering to said subject a composition comprising an antigen and anadjuvant, wherein the composition is administered to the subject by theintra-lung or pulmonary route.

Preferably, the composition is formulated to be suitable for intra-lungor pulmonary administration.

In another aspect, the present invention provides the use of acomposition comprising an antigen and an adjuvant in, or in themanufacture of a medicament for, intra-lung or pulmonary administrationto a human or animal subject to elicit or induce an immune response inthe subject.

In yet another aspect, the invention provides an agent for eliciting orinducing an immune response in a human or animal subject by theintra-lung or pulmonary route, wherein said agent is a compositioncomprising an antigen and an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIGS. 2, 3, 4, 6, 9 and 10:

-   -   Horizontal bar=median value    -   Open bar=middle 50% of values    -   Vertical line=10 and 90 percentiles.

FIG. 1 is a diagram of the sheep lung illustrating the regions whichcorrespond to upper lung and lower (or deep) lung (lung diagram modifiedfrom Nickel et al., 1979).

FIGS. 2 and 3 are graphical representations of the results obtained ininitial experiments. Groups of sheep were immunised, by the subcutaneous(s/c) or intra-lung (lung) routes with three doses, at three weekintervals, of influenza virus antigen (influenza antigen) alone or theantigen formulated with 100 μg ISCOMATRIX™ adjuvant (IMX=ISCOMATRIX™adjuvant). Blood (serum) and lung (broncho-alveolar lavage—BAL) sampleswere taken one week prior to the first dose, two weeks after the firstand one week following the second and third doses.

FIG. 2 shows antibody responses [influenza-specific endpoint titres inan Enzyme Immunoassay (EIA) after one, two and three doses (1⁰, 2⁰ and3⁰) of formulations consisting of 15 μg influenza virus antigen alone(equivalent to a monovalent dose of currently available influenzavaccines) and diminishing quantities of influenza antigen formulatedwith 100 μg ISCOMATRIX™ adjuvant. Assays for both IgG and IgA werecarried out on serum and BAL (lung) samples.

(* significant differences using Mann-Whitney analysis)

FIG. 3 shows antibody responses using the same immunisation and samplingschedule and assay procedure as for FIG. 2, where immunisation by thesubcutaneous and intra-lung routes with a very low dose of influenzaantigen (0.04 μg) with and without ISCOMATRIX™ adjuvant was carried out.

(#, *, ^, §,

significant differences using Mann-Whitney analysis)

For FIGS. 4, 6, 7, 9, 10 statistical analysis was carried out after thedata was log transformed and the groups compared by analysis of variance(ANOVA) with Dunnett's post-hoc analysis, using SPSS software version13.

FIG. 4 shows antibody responses in groups of sheep after immunisationwith three doses of 0.04 μg influenza antigen formulated with 100 μgISCOMATRIX™ adjuvant delivered to either the upper or lower lung (FIG.1). Serum and BAL (lung) samples were collected one week after the thirddose.

FIG. 5 is a Coomassie-stained SDS Polyacrylamide gel electrophoresis(SDS-PAGE) gel of purified cytomegalovirus (CMV) ΔgB protein, after theprotein was purified on an affinity column of the gB-specific 58-15monoclonal antibody coupled to an NHS-activated HP column. Lanes 1 to 4show ΔgB purified from four different affinity purification runs.

FIG. 6 shows CMV ΔgB-specific antibody responses in groups of 8 sheepafter three doses of CMV ΔgB formulated with 100 μg ISCOMATRIX™ adjuvantdelivered by either the subcutaneous or intra-lung routes. Antibodyresponses in serum and BAL samples were assayed and endpoint titresdetermined for IgG and IgA using a ΔgB-specific EIA. The graphs showantibody responses two weeks after the first dose (post-primary), andone week after the second (post-secondary) and third (post-tertiary)doses.

(* significant differences using ANOVA).

FIG. 7 shows individual percentage neutralisation titres against CMV ofindividual sheep from the same groups of sheep as FIG. 6. Serum samplesfrom bleeds taken one week prior to the first dose (Preimmune), twoweeks after the first dose (Secondary) and one week after the second(Secondary) and third (Tertiary) doses of CMV ΔgB formulated with 100 μgISCOMATRIX™ adjuvant were assayed for CMV neutralising activity. Eachcircular point is the percentage neutralisation for an individual sheepserum collected at the nominated dose point. Serum CMV-neutralisingtitres following two and three doses of the formulation weresignificantly greater than for preimmune sera for both intra-lung andsubcutaneous delivery.

(* significant differences using ANOVA).

FIG. 8 shows the results of in vitro restimulation of peripheral bloodmononuclear cells (PBMCs) following incubation with the ΔgB protein. ThePBMCs were collected from the peripheral blood of the same sheep as inExample 7, one week after administration of the third dose of CMV ΔgBformulated with 100 μg ISCOMATRIX™ adjuvant. Following stimulation withΔgB, cell proliferation was determined by assay of tritiated thymidineincorporated into the cells. Stimulation index (SI) was calculated asthe ratio of tritium incorporated into cells stimulated with ΔgB totritium incorporated into control unstimulated cells. An SI≧4 wasconsidered to be a positive proliferative response.

FIG. 9 shows antibody responses in groups of sheep (4 sheep in Flu NoAdjuvant group; 7 sheep in each of adjuvant groups), immunised withinfluenza antigen formulated with a variety of adjuvants. Theimmunisation and sampling schedules and assay procedure were the same asfor FIG. 1.

(* significant differences using ANOVA).

FIG. 10 shows haemagglutination inhibition assay (HAI) results for thesame groups of sheep as FIG. 9. HAI titre for each sample was determinedby end point inhibition on turkey red cells.

(* significant differences using ANOVA).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method of eliciting orinducing an immune response in a human or animal subject, whichcomprises administering to said subject a composition comprising anantigen and an adjuvant, wherein the composition is administered to thesubject by the intra-lung route.

Preferably, the subject is a human, however the method of the inventionalso extends to eliciting or inducing an immune response in an animalsubject such as a livestock animal (e.g., sheep, cow or horse),laboratory test animal (e.g., mouse, rat, rabbit or guinea pig),companion animal (e.g., dog or cat) or wild animal.

As used herein, references to “intra-lung” or “pulmonary” delivery of acomposition refer to delivery of the composition to mucosal surfaces ofthe lung where the active components of the composition (i.e. antigenand adjuvant) can contact the capillary system in the lung and/or themucosal immune system. These terms include “deep lung” or “lower lung”delivery of the composition, that is delivery of the composition to thedeep bronchi, bronchioli and/or alveoli of the lung.

Preferably, in accordance with this invention the composition comprisingantigen and adjuvant is administered into the lung(s) of the subject asan aerosol or in dry powder form, with the aerosol or dry powder beingdelivered using a nebuliser or similar device. Standard devices orproducts for intra-lung or pulmonary delivery of pharmaceutically activeproducts are well known to persons skilled in the art.

As indicated above, in another aspect the present invention provides theuse of a composition comprising an antigen and an adjuvant in, or in themanufacture of a medicament for, intra-lung administration to a human oranimal subject to elicit or induce an immune response in the subject.

In yet another aspect, the invention provides an agent for eliciting orinducing an immune response in a human or animal subject by theintra-lung route, wherein said agent is a composition comprising anantigen and an adjuvant.

Preferably, the antigen which is administered in accordance with thepresent invention is the antigen which will elicit or induce an immuneresponse against a lung pathogen such as influenza, Chlamydiapneumoniae, respiratory syncytial virus, pneumococci, etc. It is to beunderstood, however, that the antigen may also be selected to elicit orinduce an immune response against other pathogens, including pathogensof other mucosal sites, for example, Helicobacter pylori, Salmonella, E.coli, cholera, HIV, sexually transmitted disease organisms, etc. Antigenadministered in accordance with the present invention has the advantageof high recipient acceptability (avoidance of “needle phobia” and easeof administration). In particular, it results in both strong mucosal andsystemic immune responses, indicating that it will be useful for allvaccinations including those reliant upon a systemic immune response.

The antigen may also be a tumour-specific or tumour-associated antigen.Preferably the tumour is one associated with a mucosal site. Tumoursassociated with mucosal sites include but are not limited to lungtumours, tumours of the gastrointestinal tract and genital tracttumours.

The antigen may be any chemical entity which can elicit or induce animmune response in a human or animal, including but not limited to awhole-cell inactivated bacterium or a subunit thereof, a wholeinactivated virus or a subunit thereof, a protein or peptide, aglycoprotein, a carbohydrate, a ganglioside, a polysaccharide, alipopolysaccharide or a lipopeptide; or it can be a combination of anyof these.

Preferably also, the adjuvant is an immunostimulating adjuvant, morepreferably a saponin-based adjuvant, and even more particularly animmunostimulating complex (or ISCOM™), such as ISCOMATRIX™ adjuvant.However, the present invention also encompasses the use of otherimmunostimulating adjuvants, either individually or in combination withanother adjuvant such as an immunostimulating complex, including forexample liposomes, oil-in-water adjuvants such as MF59, aluminium saltadjuvants such as aluminium hydroxide and aluminium phosphate,lipopolysaccharide adjuvants such as lipid A and monophosphoryl lipid A(MPL), oligonucleotide adjuvants such as CpG oligonucleotide adjuvant,and mucosal adjuvants such as cholera toxin. Suitable immunostimulatingadjuvants are described by way of example by Cox and Coulter, 1997.

The composition of the invention may be delivered to the lungs using anyone of a number of existing technologies as well as those indevelopment. Numerous examples of mechanical devices able to deliverdrug or protein preparations to the lung exist and nebuliser and aerosoldevices have been used for decades in the treatment of asthma (seeGonda, 2000). Typically the devices are intended to deliver material tothe lungs by oral inhalation. Recent advances in this field (see Edwardsand Dunbar, 2002) include those made by the 3M Corporation (Shoyele andSlowey, 2006) and Inhale Therapeutics Systems, Inc. (Kuo andLechuga-Ballesteros, 2003).

In accordance with this invention, the composition is administered tothe human or animal subject in an immunologically effective amount. Asused herein, an immunologically effective amount means that amountnecessary at least partly to attain the desired immune response, or todelay the onset of, inhibit the progression of, or halt altogether, theonset or progression of the particular condition being treated. Thisamount varies depending upon the health and physical condition of theindividual to be treated, the taxonomic group of individual to betreated, the individual's immune competence, the degree of protectiondesired, the formulation of the vaccine, the assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials.

It is to be noted, however, that particularly important advantages ofthe present invention include the fact that the intra-lungadministration of the vaccine composition comprising an antigen and anadjuvant has been found to give rise not only to a systemic antibodyresponse to the antigen as well as a mucosal response, but also to thepossibility of a major reduction in the antigen dose required to elicitor induce such systemic and mucosal responses. In addition, effectiveintra-lung administration of the vaccine composition in accordance withthe present invention avoids the necessity for complex delivery systemssuch as microencapsulation or mucoadhesive or other technologies toenhance uptake of the vaccine at mucosal sites.

Furthermore, the induction of greatly improved mucosal antibodyresponses indicates that intra-lung immunisation may enhance vaccineefficacy for mucosal infections and potentially reduce pathogentransmission from an immunised/infected person.

In particular, mucosal antibodies may be of great value in improvingprotective immunity against influenza challenge. A recent WHOconsultation report (Cassetti et al., 2006) stated: “In mouse models,mucosal IgA is associated with protection against challenge and instudies with live attenuated influenza vaccines in man, the presence ofmucosal IgA correlates with reduction in virus shedding and resistanceto experimental infection. It was concluded that mucosal IgA appears toplay a role in protection against influenza virus infection and thatmore studies should evaluate the ability of vaccines to induce mucosalimmune responses . . . ”.

Accordingly, the intra-lung delivery route of the present invention isof potential value for vaccines against other infections and provides analternative route of delivery for vaccines requiring a primarilysystemic antibody response.

Furthermore, antigen which is administered in accordance with thepresent invention induces strong cellular immune responses. Antigen withadjuvant delivered by the intra-lung route stimulates the production ofperipheral blood cells which proliferate specifically in response to theantigen. These cells belong to both the CD4+ and CD8+ classes of T cellswhich have functions in enhancing antibody responses and in carrying outeffector functions such as the killing of virus-infected cells. Theability to induce cellular immune responses in addition to antibodyresponses is of potential value in vaccination against infectiousagents, particularly HIV, herpesviruses and bacteria which include anintracellular phase in their lifecycle such as mycobacteria, chlamydiaand listeria.

Further features of the present invention are more fully described inthe following Examples. It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention, and should not be understood in any way as arestriction on the broad description of the invention as set out above.

EXAMPLES Example 1 Background

By way of background to the present invention, the inventors examinedmodes of vaccine delivery in the cannulated sheep model. Cannulationallows the outflow of the local draining lymph node to be studied insome detail. This work was performed in sheep using ISCOMATRIX™ adjuvantinfluenza virus vaccine as the vaccine model. A principal finding fromthis work was that intranasal delivery was highly inefficient, inducingpoor local immunity even at high antigen doses.

These findings prompted a change in direction for the project to studythe outcome of vaccine delivery in uncannulated sheep by the intra-lungroute, by delivering vaccine deep into one lobe of the lung.

Experimental Methods

Blood Collection

Sheep were restrained and 10 mL of blood collected from the jugular veinusing a 10 mL syringe and an 18 G needle.

Immunisations

Influenza antigen used in these studies was sucrose gradient purifiedA/New Calcdonia 20/99 H1N1 virus, which had been inactivated anddetergent disrupted (Coulter et al., 1998). Antigen concentration wasbased on haemagglutinin content and determined by single radialimmunodiffusion. GMP grade ISCOMATRIX™ adjuvant was prepared by CSLLimited according to a previously described process (Pearse and Drane,2005). Prior to immunisation, vaccine formulations were prepared byadmixing an appropriate quantity of antigen with ISCOMATRIX™ adjuvant.

For intra-lung immunisations, sheep were carefully restrained in aharness and a bronchoscope inserted via the left nostril to the caudallobe of the left lung. Vaccine formulations were then infused in a totalvolume of 5 mL, followed by 10 mL of air to ensure complete delivery.

For subcutaneous immunisations, vaccine formulations were delivered in atotal volume of 200 μL in the inner thigh using a 1 mL syringe and a 25G needle.

Bronchoalveolar Lavage (BAL) Collection

For collection of bronchoalveolar lavage (BAL) samples, a bronchoscopewas inserted into restrained sheep as for vaccinations and 10 mL of PBS(phosphate-buffered saline pH7.2) delivered into the lung lobe using asyringe attached to the bronchoscope. The same syringe was then used towithdraw BAL fluid via the bronchoscope.

Evaluation of Antibody Responses by EIA

Anti-influenza antibodies in BAL and serum samples in duplicate wereevaluated by EIA (Enzyme Immunoassay). Briefly, 96 well Maxisorp flatbottom plates (NUNC, Roskilde, Denmark) were coated overnight with 50 μLof 10 μg/mL influenza antigen in carbonate buffer, pH9.6. Plates werethen blocked with 1% sodium casein before adding 100 μL of 1 in 5 serialdilutions of samples in duplicates. Binding of specific anti-influenzaantibodies was detected using rabbit anti-sheep total Ig conjugated withhorse radish peroxidase (Dako, Denmark) or anti-bovine/ovine IgA(Serotec, Oxford) followed by rabbit anti-mouse horse radish peroxidase(Dako). Colour was developed by addition of TMB substrate (Zymed, SanFrancisco), stopped by addition of 2M H₂SO₄. Optical density at 450 nmwas determined on a Bio-Tek ELx800 plate reader and antibody endpointtitres calculated.

Evaluation of Haemagglutination Inhibition Activity

Serum and BAL samples which gave an antigen-specific antibody responseby ELISA were also examined for Haemagglutination Inhibition Activity(HAI). This assay determines the titre of functional antibodies, bymeasuring the inhibition of red blood cell agglutination by influenzavirus. Samples were tested for HAI against egg grown A/NewCalcdonia/20/99 virus (H1N1) using turkey red blood cells. The HAI titrewas determined as the endpoint dilution that inhibited influenzaagglutination of the red blood cells. HAI assays were performed by theWHO Collaborating Centre for Reference and Research on Influenza,Melbourne, Australia.

In Vitro Antigen Restimulation of Peripheral Blood Mononuclear Cells

Blood samples (50 mL) collected from the jugular vein using a syringeand 18 G needle were placed into a 50 mL tube containing 100 μL of 5000U/mL heparin. The cells were then centrifuged at 800 g for 20 minuteswith no brake, the buffy coat collected into a 15 mL tube and diluted to8 mL with PBS. Using a transfer pipette, 3.5 mL of Ficoll-paque wasadded to the bottom of the cell suspension, and the samples centrifugedat 1000 g for 30 minutes with no brake. The peripheral blood mononuclearcells were then collected from the Ficoll-PBS interface, washed in PBSand resuspended at 5×10⁶/mL in complete medium (Dulbecco's ModifiedEagles medium supplemented with 10% FCS, L-glutamine, penicillin (100U/mL), streptomycin (100 μg/mL) and 50 μM 2-mercaptoethanol).

For the antigen restimulation assay, 100 μL of cells (5×10⁵/well) werealiquoted into 96-well tissue culture plates, to which were added intriplicate 100 μL of either media alone or media containing 10 or 20μg/mL A/New Calcdonia influenza antigen (final concentration 5 or 10μg/mL antigen). After 5 days culture at 37° C. in a humidifiedincubator, all wells were pulsed with 20 μL of 1 μCi tritiated thymidinefor 24 hours. Cells were then harvested onto glass fibre filters using aPackard Harvester, placed into cassettes with Microscint scintillationliquid, and β-radiation measured using an automated microplatescintillation counter.

Evaluation of Immunisation to the Upper and Lower Lung

Examination of the effectiveness of immunisation by delivery to theupper lung, as an alternative to delivery to the lower lung was carriedout as described below. FIG. 1 (from Nickel et al., 1979) illustratesthe sites of the sheep lung used for delivery to the upper and lowerlung.

Vaccine delivered to the ‘upper lung’—the vaccine was delivered into theupper lung using a fibre-optic bronchoscope. The vaccine was introducedinto the principle bronchi, 1 cm past the major tracheal bifurcation.

Vaccine delivered to the ‘lower lung’—the vaccine was delivered deepinto the caudal lung using a fibre-optic bronchoscope. The vaccine wasintroduced where the caudal bronchus joins the caudal segmental bronchi,10 cm past the major tracheal bifurcation.

Immunisations, bleeds, BAL collection and ELISA assays were carried outas described for the other experiments. The sheep received three dosesof 0.04 μg influenza antigen with 100 μg ISCOMATRIX™ adjuvant.

Results

Antibody Induction by Immunisation Via the Intra-Lung Route

In 3 separate experiments sheep were immunised by the intra-lung route.Bleeds were taken at 2 weeks post 1° dose, and one week post 2° and 3°doses

Experiment 1 Sheep were immunised with either 1, 5 or 15 μg of antigenplus 100 μg ISCOMATRIX™ adjuvant. Three immunisations were given, 3weeks apart. Serum samples were collected before commencement, and aftereach vaccination.

Following the surprising finding that 1 μg of adjuvant influenza antigendelivered intra-lung was as effective as 15 μg, a second experimentexamined further reductions in antigen dose.

Experiment 2 Sheep were immunised with either 0.04, 0.2, 1, 5 or 15 μgantigen plus 100 μg ISCOMATRIX™ adjuvant. Three immunisations weregiven, 3 weeks apart. Serum samples were collected before commencement,and after each immunisation.

These experiments demonstrated that all antigen doses inducedsignificant antibody responses in recipient animals—even as low as 0.04μg antigen. The lower antigen doses induced very few antibodies afterone dose. However, immunity in recipient animals was equivalent to thehigher antigen doses following 2 and 3 doses. These antibody responsesincluded functional haemagglutination inhibition (HAI).

Serum IgG and IgA levels in sheep immunised intra-lung with 0.04 μgantigen and 100 μg ISCOMATRIX™ adjuvant were equivalent to thoseobtained following subcutaneous immunisation with 15 μg of antigen alone(current vaccine dose). The low dose intra-lung immunisations producedvery good levels of specific IgA and IgG in the lung (BAL—bronchialalveolar lavage), superior to that induced by the higher doses injectedsubcutaneously.

Experiment 3 The third experiment evaluated (i) the requirement foradjuvant for inducing significant immunity with low antigen dose, (ii)directly compared low antigen dose (0.04 μg) delivered intra-lung withlow dose delivered subcutaneously, (iii) examined even lower antigendoses (0.008 μg antigen).

The third experiment:

-   (a) repeated the observations made in the first two;-   (b) demonstrated that adjuvant was essential for the induction of    immunity against very low dose antigen;-   (c) found that adjuvanted low dose antigen delivered intra-lung    induced similar serum antibodies and superior lung antibodies    compared to the same low antigen dose vaccine or the current vaccine    dose, delivered subcutaneously;-   (d) found that more than 2 doses of adjuvanted very low dose antigen    injected subcutaneously appeared to induce a tolerance or inhibitory    effect as serum antibodies following a 3rd dose were reduced. This    inhibition did not occur following lung delivery;-   (e) found that intra-lung immunisation with even 0.008 μg antigen    administered with 100 μg ISCOMATRIX™ adjuvant induced significant    antibody induction, though less than induced by 0.04 μg antigen with    ISCOMATRIX™ adjuvant.

The combined data for experiments 1 to 3 are summarised in FIG. 2 andTable 1.

TABLE 1 Haemagglutination Inhibition (HAI) activity induced byintra-lung immunisation with reducing doses of influenza antigen andISCOMATRIX ™ adjuvant Median HAI titre (Interquartile range) SerumBronchoalveolar lavage Route Vaccine Primary Secondary Tertiary PrimarySecondary Tertiary S/C 15 μg flu 5 30 100 0 0 0 (0-18) (20-100) (20-440)(0) (0) (0) Lung 15 μg flu + 10  330   60 0 10* 10* IMX (10)  (20-640)(35-400) (0) (10-20) (10-20)  Lung 5 μg flu + 0 160  240 0 0 40* IMX (0)(120-160)  (70-400) (0)  (0-10) (8-50) Lung 1 μg flu + 0 30 160 0 0 5IMX (0) (15-40)  (140-320)  (0) (0) (0-10) Lung 0.2 μg flu + 0 15 160 00 5 IMX (0) (0-40) (65-200) (0) (0) (0-13) Lung 0.04 μg flu + 0 10 160 00 30* IMX (0) (0-40) (140-640)  (0) (0) (0-80) Lung 0.008 μg 0  0  20 00 0 flu + IMX (0)  (0) (0-40) (0) (0) (0) Table 1 legend: Data iscombined from 3 separate experiments. Groups of sheep (n = 8 per group,except the 15 μg flu group (n = 12) and the 0.04 μg flu + IMX group (n =16)) were immunised in the left lung with reducing doses of split virioninfluenza antigen plus 100 μg of ISCOMATRIX ™ adjuvant (IMX). A group ofvaccine control sheep were immunised subcutaneously (S/C) with anequivalent dose of one strain of the current human influenza vaccine (15μg influenza antigen alone). Sheep received 3 immunisations, spaced by 3weeks. Sera and left lung washings (BAL) were collected from all sheepone week prior to and two weeks after the primary dose and one weekafter the secondary and tertiary doses. The titration of these sampleswhich inhibited influenza mediated haemagglutination was thendetermined. All preimmunisation samples were negative for HAI activity(not shown). *Significantly higher HAI titre compared withsubcutaneously immunised control (Mann Whitney; p < 0.004).

The data for experiment 3 are shown in Table 2 and FIG. 3.

TABLE 2 Requirement of adjuvant and route of delivery onHaemagglutination Inhibition activity induced by immunisation withextremely low doses of influenza antigen Median HAI titre (Interquartilerange) Serum Bronchoalveolar lavage Route Vaccine Primary SecondaryTertiary Primary Secondary Tertiary S/C 15 μg flu 0 30 30 0 0 0 (0-3)  (20-100) (18-160) (0) (0) (0) S/C 0.04 μg 0 10 25 0 0 0 flu (0) (0-40)(8-50) (0) (0) (0) S/C 0.04 μg 20*{circumflex over ( )} 320*{circumflexover ( )} 160{circumflex over ( )} 0 0 0 flu + IMX (8-20) (280-400)(80-320) (0) (0) (0) Lung 0.04 μg 0  0#  0# 0 0 0 flu (0)  (0)  (0) (0)(0) (0) Lung 0.04 μg 0  5 160{circumflex over ( )} 0 0 60*{circumflexover ( )} flu + IMX (0)  (0-18) (80-200) (0) (0) (30-100) Table 2legend: Groups of sheep (n = 8) were immunised in the left lung with anextremely low dose (0.04 μg) of split virion influenza antigen with orwithout 100 μg of ISCOMATRIX ™ adjuvant (IMX). Other groups received thesame vaccines injected subcutaneously (S/C). One group of control sheepwas immunised subcutaneously with an equivalent antigen dose of onestrain of the current human influenza vaccine (15 μg influenza antigenalone). Sheep received 3 immunisations, spaced by 3 weeks. Sera and leftlung washings (BAL) were collected from all sheep one week prior to andtwo weeks after the primary immunisation and one week after thesecondary and tertiary doses. The titration of these samples whichinhibited influenza mediated haemagglutination was then determined. Allpreimmunisation samples were negative for HAI activity (not shown).#Significantly lower HAI titre compared with subcutaneously immunisedcontrol (Mann Whitney; p < 0.002). *Significantly higher HAI titrecompared with subcutaneously immunised control (Mann Whitney; p <0.028). {circumflex over ( )}Significantly higher HAI titre comparedwith unadjuvanted group (Mann Whitney; p < 0.01).

In FIGS. 1 and 2:

-   -   Horizontal bar=median value    -   Open bar=middle 50% of values    -   Vertical line=10 and 90 percentiles.    -   IMX=ISCOMATRIX™ adjuvant

The bars show the influenza-specific endpoint antibody titre after thefirst (1⁰), second (2⁰) and third (3⁰) immunisations as described above.

In FIG. 1: *Significantly greater than 15 μg s/c group (Mann-Whitney;p<03).

In FIG. 2

-   -   # Significantly less than 15 μg s/c group (Mann-Whitney;        p<0.01).    -   * Significantly greater than 15 μg s/c group (Mann-Whitney;        p<0.038).    -   ^ Significantly greater than unadjuvanted vaccine delivered via        the same route (Mann-Whitney; p<0.038).    -   §Subcutaneous, significantly greater than same vaccine delivered        intralung (Mann-Whitney; p<0.028).    -   Intralung, significantly greater than same vaccine delivered        subcutaneously (Mann-Whitney; p<0.021).

Experiment 4 This experiment evaluated the effectiveness of delivery ofadjuvanted influenza antigen to the upper lung, in comparison withdelivery to the lower lung used in earlier experiments. Two groups of 8sheep received a formulation containing 0.04 μg influenza antigen and100 μg ISCOMATRIX™ adjuvant to either the upper or lower lung threetimes and serum and BAL antibody responses were examined by EIA (FIG.4).

These results indicated that after three doses, although delivery to theupper lung induced serum and BAL responses, significantly betterresponses in both serum and BAL for both IgG and IgA were induced bylower lung delivery.

Summary

Intra-lung immunisation with influenza virus antigen with 100 μgISCOMATRIX™ adjuvant proved highly efficient in the induction of bothsystemic and mucosal (BAL) antibody responses with HAI activity.

Strong serum antibody responses were observed even at extremely lowlevels of antigen (0.04 μg influenza antigen with ISCOMATRIX™ adjuvant).These responses were much stronger than those induced by subcutaneousinjection (s/c) of 15 μg of influenza antigen alone (as per the currentvaccine) and matched those induced by subcutaneous 0.04 μg influenzaantigen with ISCOMATRIX™ adjuvant.

The mucosal antibody response induced by intra-lung delivery of 0.04 μginfluenza antigen with ISCOMATRIX™ adjuvant was greatly elevatedcompared with subcutaneous 15 μg of influenza antigen or subcutaneous0.04 μg influenza antigen with ISCOMATRIX™ adjuvant. Neither of thelatter induced detectable mucosal (lung) responses.

Example 2 Cellular Immunity Induction by Vaccination Via the Intra-LungRoute

Sheep received 4 intra-lung immunisations with either 0.04 μg influenzaantigen alone, or 0.04 μg influenza+100 μg ISCOMATRIX™ adjuvant. Oneweek after the last immunisation, peripheral blood was collected, andmononuclear cells cultured in 96 well plates with either media alone(negative control), or 5 or 10 μg influenza antigen (restimulated).After five days, wells were pulsed with tritiated thymidine for 24hours. Radioactivity was then measured to enumerate cell proliferation.The Stimulation Indices were calculated by dividing the mean counts perminute (cpm) for the restimulated group by the mean cpm for the mediumalone control group.

The results of a cell proliferation study are shown in Table 3. Whensheep were vaccinated intra-lung with antigen alone, no proliferativeresponse was detectable in the peripheral blood. However, such aresponse was detected in sheep which received antigen with ISCOMATRIX™adjuvant. These data demonstrate the requirement for adjuvant, followinglung vaccine delivery, to induce a cellular proliferative memoryresponse in the peripheral circulation.

TABLE 3 Proliferative response of influenza-stimulated peripheral bloodmononuclear cells, from sheep immunised intra-lung with low doseantigen, with or without ISCOMATRIX ™ adjuvant Median Stimulation Index(Interquartile Range) following restimulation with 5 μg 10 μg Intra-lungvaccination influenza influenza Vaccine Group size antigen antigen 0.04μg influenza n = 8 1 (1-2)  1 (1-3) 0.04 μg influenza + n = 8 6 (2-16)10 (3-25) 100 μg ISCOMATRIX ™ adjuvant

Example 3 Formulation of Antigen with Microfluidised Oil-in-WaterAdjuvant for Administration by the Intra-Lung Route

Antigen at an appropriate concentration (to result in a final antigenconcentration yielding a single dose in the range 0.01 to 500 μg) ismixed with 5% v/v Squalane, 0.5% v/v Tween 80, 0.5% v/v Span 85 in PBSpH 7.2, 0.05% sodium azide. This mixture is then subjected to highpressure microfluidisation at a pressure of 19-20,000 pounds per squareinch (psi). The microfluidisation is repeated a further seven times. Theformulation is delivered via the intra-lung route by the methodsdescribed above.

Example 4 Systemic and Mucosal Immunity Induced by Immunisation Via theIntra-Lung Route with Recombinant Cytomegalovirus gB Antigen Formulatedwith ISCOMATRIX™ Adjuvant

In order to confirm and extend observations made with influenza antigenand ISCOMATRIX™ adjuvant, studies were performed in sheep examining theimmune responses induced by intra-lung delivery of a recombinant viralantigen. A truncated form of cytomegalovirus gB glycoprotein (CMV gB)formulated with ISCOMATRIX™ adjuvant was delivered by the intra-lung andsubcutaneous routes and antibody responses in serum and lung assessed byEIA. In addition, induction of functional antibodies was assessed byassay of CMV-neutralising antibodies in serum. The ability offormulations delivered intra-lung to induce T cell responses was alsoexamined by assessing gB-specific cell proliferation of peripheral bloodmononuclear cells

Experimental Methods

Production of Truncated Recombinant Human CMV gB Protein (ΔgB)

1. Generation of Plasmid Containing DNA Encoding ΔgB Protein.

DNA encoding a truncated form of CMV gB which had been engineered toremove the transmembrane region of the protein was provided by Assoc.Prof. Rajiv Khanna, Queensland Institute of Medical Research, Brisbane,Australia. This DNA encoded a protein containing the signal sequence ofTissue Plasminogen Activator (TPA) with the ecto- and cytoplasmicdomains (NCBI P06473) of gB fused in frame. As well, the DNA was pointmutated at the nucleotides encoding the region around amino acids460/461 to prevent cleavage of the expressed protein. The DNA was clonedinto the pCEP4 vector (Invitrogen) as a KpnI/NotI fragment. Theresulting plasmid (pCEP4ΔgB) was amplified in E. coli and purifiedplasmid prepared using Qiagen Maxi Kit.

2. Expression of ΔgB Protein in Mammalian Cells

Cultures of FreeStyle™ 293-F cells grown in FreeStyle™ Expression Medium(Invitrogen) in shake flasks at 37° C. were transfected with pCEP4ΔgBusing 293fectin transfection reagent (Invitrogen). Secreted expressionof the protein ΔgB under selection using Hygromycin B in the cultureswas determined by SDS-PAGE gel analysis of cell supernatant samples.Cell culture supernatants were harvested and clarified by centrifugationat 2500 rpm and then passed through a 0.45 μm filter prior tochromatography.

3. Purification of ΔgB Protein

The ΔgB protein was purified using affinity chromatography by capturewith the gB-specific monoclonal antibody 58-15 (Singh and Compton,2000). The antibody was purified on a Prosep-A (Millipore) protein Acolumn. For purification of ΔgB protein, the purified antibody wascoupled to a HiTrap NHS-activated HP column (Pharmacia-Amersham) inequilibration buffer (0.2M NaHCO₃/0.5M NaCl, pH8.3). Clarified cellculture supernatant was diluted 1:1 with 100 mM Borate/150 mM NaClpH8.5. The diluted supernatant was applied to the HiTrap column,equilibrated with equilibration buffer and eluted firstly with 0.1MGlycine-HCl pH2.5, followed by equilibration buffer and eluted again,this time with 0.15M Ammonium Hydroxide pH10.5. Eluted fractions wereneutralised using either 3M Tris pH8 or 1M Glycine pH2.5. Protein puritywas examined by Coomassie-stained SDS-PAGE gel electrophoresis (FIG. 5).Protein concentration was estimated by determining the OD at 280 nm.

Immunisation of Sheep with ΔgB

Blood Collection

Methods were the same as those described in Example 1.

Immunisations

Groups of sheep were immunised with ΔgB protein formulated withISCOMATRIX™ adjuvant Table 4). ΔgB protein from cytomegalovirus wasprepared as described.

All sheep received three vaccine doses, the details of which, includingthe blood collection schedule are described in Example 1.

TABLE 4 Groups of sheep were immunised by the indicated route with ΔgBprotein formulated with ISCOMATRIX ™ adjuvant. Route of Group Sizedelivery Antigen Adjuvant 1 n = 8 Subcutaneous 15 μg CMV ΔgB 100 μgISCOMATRIX ™ adjuvant 2 n = 8 Intra-lung 15 μg CMV ΔgB ISCOMATRIX ™adjuvantBAL Collection

Collection of BAL samples was performed as described in Example 1.

Evaluation of Antibody Responses by EIA

Methods for detecting sheep IgG and IgA were similar to those in Example1 with the following exceptions. Plates (96 well) were coated overnightwith 100 μl per well of 2 μg/mL purified ΔgB protein in PBS. Bound sheepIgG was detected with rabbit anti-sheep IgG (H+L) conjugated to horseradish peroxidise (Southern Biotech). In the case of both IgG and IgATMB peroxidise substrate was sourced from KPL Kirkegaard and PerryLaboratories respectively. The reaction was stopped by addition of 50 μlper well 0.5M H₂SO₄.

Evaluation of Functional Activity Against CMV

The capacity of the vaccines to induce functional serum antibodies wasassessed using a neutralisation assay based on recombinant humancytomegalovirus infection of MRC-5 cells. Assays were performed byAssoc. Prof. Rajiv Khanna and colleagues, Queensland Institute ofMedical Research, Brisbane, Australia, using recombinant CMV Ad169wt86-EGFP expressing green fluorescent protein (Marschall et al., 2000;Wang et al., 2004). After exposure of Ad169wt86-EGFP to serum dilutions,the ability of the virus to infect human fibroblasts (MRC-5 cells) wasassessed by quantifying the percentage cell nuclei fluorescing usingflow cytometry (FACS). The procedure is described below:

-   -   1. Sera from the sheep were incubated at 56° C. for 30 minutes.    -   2. Sera from the sheep (neat and at 2-fold and 4-fold dilutions        in 10041 DMEM) were dispensed in a 96 well tray.    -   3. 25 μl (2.5×10⁴ PFU) Ad169wt86-EGFP virus was added to each        serum sample.    -   4. Serum/virus samples were incubated at 37° C. for 2 hours in        7% CO₂ in air.    -   5. Previously, 48 well flat-bottom trays containing MRC-5 cells,        grown on DMEM with 10% foetal bovine serum (DMEM-10) by        incubation at 37° C. in 7% CO₂ in air, had been prepared and        were at 80-90% confluency at time of use.    -   6. The cell culture medium was removed from the MRC-5 cells. 50        μl of each virus/serum sample was then transferred to        corresponding wells in the 48 well flat-bottom trays containing        MRC-5 cells, 50 μl DMEM added and the 48 well trays incubated at        37° C. for 2 hours in 7% CO₂ in air with gentle rocking.    -   7. The inoculum was aspirated from the 48 well trays, the wells        washed three times with DMEM-10, and then 500 μl DMEM-10 added        to each well, and incubated at 37° C. in 7% CO₂ in air        overnight.    -   8. The next day the cell medium and cells (using trypsin) were        harvested from the 48 well trays and the cells pelleted by        centrifugation.    -   9. The pellets were resuspended in PBS and pelleted again by        centrifugation.    -   10. The pellets were resuspended vigorously in 100 μl of        hypotonic buffer (0.1% sodium citrate, 0.1% (v/v) Triton X-100)        containing 0.2 μl mg/mL 7-Aminoctinmycin D and left for 20        minutes at room temperature in the dark.    -   11. Samples of each pellet were transferred to tubes suitable        for FACS analysis and PBS containing 1% Paraformaldehyde added.        The mixture was left on ice for 30 minutes prior to FACS        analysis.    -   12. The samples were analysed on a Becton Dickinson FACSCanto        and the percentage of fluorescing nuclei in the samples        quantified.    -   13. The percentage neutralisation for each sample was calculated        using the formula:        % neutralisation=[percentage of fluorescing nuclei (no serum        added)−percentage of fluorescing nuclei (serum        sample)−percentage of fluorescing nuclei (no serum added)]×100.        In Vitro Antigen Restimulation of Peripheral Blood Mononuclear        Cells

Blood samples (50 mL) collected from the jugular vein using a syringeand 18 G needle were placed into a 50 mL tube containing 100 μL of 5000U/mL heparin. The cells were then centrifuged at 800 g for 20 minuteswith no brake, the buffy coat collected into a 15 mL tube and diluted to8 mL with PBS. Using a transfer pipette, 3.5 mL of Ficoll-paque wasadded to the bottom of the cell suspension, and the samples centrifugedat 1000 g for 30 minutes with no brake. The peripheral blood mononuclearcells were then collected from the Ficoll-PBS interface, washed in PBSand resuspended at 5×10⁶/mL in complete medium (Dulbecco's ModifiedEagles medium supplemented with 10% FCS, L-glutamine, penicillin (100U/mL), streptomycin (100 μg/mL) and 50 μM 2-mercaptoethanol).

For the antigen restimulation assay, 100 μL of cells (5×10⁵/well) werealiquoted into 96-well tissue culture plates, to which were added intriplicate 100 μL of either media alone or media containing 5, 10, 20 or40 μg/mL gB protein (final concentration 2.5, 5, 10 or 20 μg/mL). After5 days culture at 37° C. in a humidified incubator, all wells werepulsed with 20 μL of 1 μCi tritiated thymidine for 24 hours. Cells werethen harvested onto glass fibre filters using a Packard Harvester,placed into cassettes with Microscint scintillation liquid, andβ-radiation measured using an automated microplate scintillationcounter.

Results

Immune Responses Following Intra-Lung and Subcutaneous Immunisation withRecombinant CMV ΔgB Antigen Formulated with ISCOMATRIX™ Adjuvant

Sheep received three subcutaneous or intra-lung doses, at 3 weekintervals of the CMV ΔgB antigen formulated with ISCOMATRIX™ adjuvant.Sera and BAL samples were collected one week prior to the first dose, 2weeks after the first dose and 1 week after the second and third doses.For antibody EIA data, preimmunisation antibody titres were subtracted.

The results of these experiments with ΔgB formulated with ISCOMATRIX™adjuvant (FIG. 6) were broadly similar to those shown in Example 2 usinglow dose influenza antigen (0.04 μg) formulated with ISCOMATRIX™adjuvant, where serum IgG and IgA responses following intra-lung wereequivalent to those for subcutaneous delivery after three doses.However, in contrast to the results obtained with influenza antigenformulations, ΔgB formulated with ISCOMATRIX™ adjuvant delivered by theintra-lung route induced equivalent serum IgG and IgA to subcutaneousdelivery after only two doses and a significantly higher serum IgG whenresponses after one dose were examined (FIGS. 6 a and b).

In the case of antibody responses in the lung, responses followingimmunisation with ΔgB formulated with ISCOMATRIX™ adjuvant againparalleled observations made with low dose influenza antigen formulatedwith ISCOMATRIX™ adjuvant. ΔgB formulated with ISCOMATRIX™ adjuvantinduced significantly higher antibody responses in the lung after threedoses when delivered intra-lung when compared to subcutaneous deliveryof the same formulation (FIGS. 6 c and d).

In the case of functional antibody induction, the results for the CMVneutralisation assays on sera were in agreement with those obtained inthe EIA for serum IgG responses (FIG. 7). Neutralising antibodyresponses induced by both intra-lung and subcutaneous delivery of ΔgBformulated with ISCOMATRIX™ adjuvant were significantly higher than inthe preimmunisation sera. The neutralising responses increased furtherafter three doses. The results indicate equivalence in induction ofneutralising antibodies in serum when antigen formulated withISCOMATRIX™ adjuvant was delivered by the intra-lung and subcutaneousroutes.

The results from assay of restimulation of peripheral blood mononuclearcells with ΔgB (FIG. 8), indicate that antigen formulated withISCOMATRIX™ adjuvant delivered by either the intra-lung or subcutaneousroutes induced T cell responses after two to three doses (stimulationindex of 4 or greater). The two routes of delivery induced equivalent Tcell responses.

Example 5 Systemic and Mucosal Immunity Induced by Vaccination Via theIntra-Lung Route with Different Adjuvant Technologies

These studies were performed to examine whether adjuvants other thanISCOMATRIX™ adjuvant were able to induce antigen-specific mucosal andsystemic antibody responses when delivered by the intra-lung route.Groups of sheep were immunised intra-lung with influenza antigenformulated with several alternative adjuvants and the responses comparedwith an ISCOMATRIX™ adjuvant formulation (Table 5).

TABLE 5 Groups of sheep immunised by the intra-lung route with influenzaantigen formulated with various adjuvant formulations Influenza antigenGroup Size dose (μg) Adjuvant 1 n = 4 15 — 2 n = 7 15 100 μgISCOMATRIX ™ adjuvant 3 n = 7 15 100 μg ISCOMATRIX ™ adjuvant + 50 μgMPL 4 n = 7 15 1 mg liposomes + 50 μg MPL 5 n = 7 15 1 mg liposomes +750 μg CpGExperimental MethodsPreparation of Adjuvant FormulationsA. Liposomes were Prepared by the Following Method:Buffer Solutions:

-   -   IR buffer: 0.14M NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 0.05 mM        CaCl₂.2H₂O, 1.5 mM KH₂PO₄ pH7.2    -   IVD buffer: 0.14M NaCl, 3.5 mM Na₂HPO₄, 1.4 mM NaH₂PO₄.2H₂O        pH7.2    -   1. A solution of 11.4 mg/mL cholesterol, 12.4 mg/mL        dipalmitoylphosphatidylcholine (DPPC) and Mega10 was prepared in        IR buffer (Chol/DPPC).    -   2. Chol/DPPC was diluted 1/40 gradually over 30 minutes by        addition of IVD buffer.    -   3. The diluted solution was concentrated by ultrafiltration at        40° C. using an ST 200 Diafiltration Cartridge (Nephral) to 1/40        of the original diluted volume.    -   4. The concentrated solution was washed by ultrafiltration with        25 volumes of IVD buffer.    -   5. The Chol/DPPC solution was concentrated 2 fold by further        ultrafiltration.    -   6. Prior to lipid extrusion Chol/DPPC was incubated at 40° C.        for at least 1 hour.    -   7. Liposomes were extruded from Chol/DPPC using a lipid extruder        (T.001 Northern Lipids Inc., Canada) fitted with a 10 mL        Thermobarrel Extruder maintained at 42° C. throughout the        procedure.    -   8. Extrusion was carried out on 10 mL lots of Chol/DPPC using a        pressure of 1400-2000 kPa through a 0.1 μm PC disc filter.    -   9. The process was repeated until all of the Chol/DPPC solution        had been subjected to 10 passes through the system.    -   10. The resulting liposomes were assayed for cholesterol and        DPPC content and examined under the electron microscope using        negative staining to assess size and homogeneity.        B. Preparation of MPL

Lipid A monophosphoryl (MPL) (Sigma L6895) was resuspended in DMSO(Sigma D2650) at 2 mg/mL and maintained at 4° C. until use.

C. Preparation of CpG

CpG (Sigma-Genosys 1062 4856-011) was resuspended in 10 mM Tris/1 mMEDTA pH8 and maintained at 4° C. until use.

D. Preparation of Adjuvant Formulations

All adjuvant formulations were prepared by mixing the relevantcomponents using PBS as diluent prior to the addition of Influenzaantigen (Table 5). Following this, the formulations containing liposomeswere subjected to sonication in a VirSonic sonicator at setting 4 usinga ⅛ inch probe. The formulations received two 5 second sonicationsseparated by 30 seconds, all performed on ice.

Immunisations

Groups of sheep were immunised with the influenza antigen and adjuvantformulations described in Table 5, and bleeds taken, using the methodsdescribed in Example 1.

BAL Collection

Collection of BAL samples was performed as described in Example 1.

Evaluation of Antibody Responses by ELISA

Assay of antibody responses to influenza antigen in serum and BALsamples was performed as described in Example 1.

Evaluation of Haemagglutination Inhibition Activity

Assay of HAI activity in serum and BAL samples was performed asdescribed in Example 1.

Results

Evaluation of Immune Responses Following Intra-Lung Vaccination withFormulations of Influenza Antigen with a Variety of Adjuvants.

Sheep received three doses at 3 week intervals. Sera and lung (BAL)samples were collected prior to and 2 weeks after the first dose, and 1week after the second and third doses. For antibody ELISA data,pre-immunisation antibody titres were subtracted.

The results of these experiments demonstrated that different adjuvantformulations varied in their ability to induce antibody responses whencombined with antigen and delivered by the intra-lung route (FIGS. 9 and10).

All adjuvants conferred some benefit in comparison with antigen alone inthe induction of antibody in serum. Formulations containing ISCOMATRIX™adjuvant induced significantly better responses than antigen alone afterthree doses in both serum and BAL and in the case of serum IgG inducedsuperior responses after two doses. Of particular note is the datashowing induction of functional (HAI) antibodies by bothISCOMATRIX™adjuvant formulations in serum after two doses and in BALafter three doses (FIG. 10).

The data (FIG. 9) also suggests an effect from the addition of MPL toISCOMATRIX™ adjuvant formulations, in that significant increases overadjuvant alone in serum IgG and IgA antibodies occurred after one andtwo doses respectively, in sheep immunised with the formulationcontaining antigen and ISCOMATRIX™ adjuvant plus MPL.

REFERENCES

-   Casetti, M. C., et al. Report of a consultation on role of    immunological assays to evaluate efficacy of influenza vaccines.    Initiative for Vaccine Research and Global Influenza Programme,    World Health Organization, Geneva, Switzerland, 25 Jan. 2005.    Vaccine (2006), 24(5):541-543.-   Coulter, A. et al. Studies on experimental adjuvanted influenza    vaccines: comparison of immune stimulating complexes (Iscoms™) and    oil-in-water vaccines. Vaccine (1998), 16 (11/12): 1243-1253.-   Cox, J. C. and Coulter, A. R. Adjuvants—a classification and review    of their modes of action. Vaccine (1997), 15(3):248-256.-   Edwards, D. A. and Dunbar, C. Bioengineering of therapeutic    aerosols. Annu Rev Biomed Eng (2002), 4:93-107.-   Gonda, I. The ascent of pulmonary drug delivery. J Pharm Sci (2000),    89:940-5-   Griffith, G. D., et al. Liposomally-encapsulated ricin toxoid    vaccine delivered intratracheally elicits a good immune response and    protects against a lethal pulmonary dose of ricin toxin. Vaccine    (1997), 15 (17/18): 1933-1939.-   Kuo, M. and Lechuga-Bellesteros, D. U.S. Pat. No. 6,518,239.-   Marschall, M., et al. Recombinant green fluorescent    protein-expressing human cytomegalovirus as a tool for screening    antiviral agents. Antimicrobial Agents and Chemotherapy (2000), 44    (6): 1588-1597.-   Nickel, R., Schummer A. and Seiferle E. Eds. The Viscera of the    Domestic Animals. 2^(nd) edition. Verlag Paul Parey, Berlin (1979).-   Pearse, M. J. and Drane, D. ISCOMATRIX™ adjuvant for antigen    delivery. Adv. Drug. Del. Rev. (2005), 57:465-474.-   Oya Alpa, H., et al. Biodegradeable mucoadhesive particulates for    nasal and pulmonary antigen and DNA delivery. Advanced Drug Delivery    Reviews (2005), 57:411-430.-   Shoyele, S. A. and Slovey, A. Prospects for formulating    proteins/peptides as aerosols for pulmonary drug delivery.    International Journal of Pharmaceutics (2006), 314:1-8.-   Singh J. and Compton T. Characterisation of a panel of insertion    mutants in human cytomegalovirus glycoprotein B. Journal of    Virology (2000) 74: 1383-1392.-   Wang, Z., et al Development of an efficient fluorescence-based    microneutralisation assay using recombinant human cytomegalovirus    strains expressing green fluorescent protein. Journal of Virological    Methods (2004), 120: 207-215.

The invention claimed is:
 1. A method for eliciting or inducing animmune response in a human or animal subject, which comprisesadministering to said subject by an intra-lung route a compositioncomprising: as the only antigen component, an antigen selected from thegroup consisting of protein antigens, peptide antigens, and polypeptideantigens, and, as the only adjuvant component(s), one or more adjuvantsselected from the group consisting of saponin-based adjuvants, aluminiumsalt adjuvants, lipopolysaccharide adjuvants. and oligonucleotideadjuvants.
 2. The method of claim 1, wherein the composition isformulated for intra-lung administration.
 3. The method of claim 2,wherein the composition is an aerosol or in dry powder form.
 4. Themethod of claim 1, wherein the composition is delivered to the subjectby oral inhalation.
 5. The method of claim 1, wherein the composition isdelivered to the lower lung of the subject.
 6. The method of claim 1,wherein the one or more adjuvants comprises an immunostimulatingadjuvant.
 7. The method of claim 6, wherein the one or more adjuvantscomprises an immunostimulating complex.
 8. The method of claim 7,wherein the one or more adjuvants comprises an immunostimulatingmolecule comprising a saponin, cholesterol and phospholipid.
 9. Themethod of claim 6, wherein the one or more adjuvants comprises animmunostimulating complex in combination with another immunostimulatingadjuvant.
 10. The method of claim 9, wherein the one or more adjuvantscomprises an immunostimulating complex in combination with alipopolysaccharide adjuvant.
 11. The method of claim 10, wherein the oneor more adjuvants comprises an immunostimulating molecule comprising asaponin, cholesterol and phospholipid in combination with monophosphoryllipid A (MPL).
 12. The method of claim 1, wherein the antigen is anantigen which elicits or induces an immune response against a lungpathogen.
 13. The method of claim 12, wherein the lung pathogen isinfluenza virus, Chlamydia pneumoniae respiratory syncytial virus orpneumococci.
 14. The method of claim 1, wherein the antigen is anantigen which elicits or induces an immune response against a pathogenof non-lung mucosal sites.
 15. The method of claim 14, in which thepathogen is Helicobacter pylori; Salmonella E. coli, cholera, HIV, or asexually transmitted disease organism.
 16. The method of claim 1,wherein the antigen is a tumour specific or tumour associated antigen.17. The method of claim 16, wherein the tumour is associated with amucosal site.
 18. The method of claim 17, wherein the tumour is a lungtumour, a tumour of the gastrointestinal tract, or a genital tracttumour.
 19. The method of claim 1, wherein the adjuvant is asaponin-based adjuvant.